CHLOROPROPANOLS
First draft prepared by
Dr P. Olsen
Institute of Toxicology, National Food Agency of Denmark
Ministry of Health, Soborg, Denmark
1. EXPLANATION
Certain chlorinated propanols occur as contaminants in
hydrolyzed vegetable proteins. The two substances considered by the
Committee at its present meeting were 3-chloro-1,2-propanediol and
1,3-dichloro-2-propanol, neither of which has previously been
evaluated by the Committee. Processing of defatted vegetable
proteins by traditional hydrochloric acid hydrolysis leads to the
formation of significant amounts of 3-chloro-1,2-propanediol and
1,3-dichloro-2-propanol. However, manufacturing techniques have
been improved, enabling the reduction of the level of 3-chloro-1,2-
propanediol to less than 2 mg/kg and that of 1,3-dichloro-2-propanol
to less than 0.02 mg/kg in hydrolyzed vegetable proteins.
Because this monograph covers the data considered by the
Committee on both 3-chloro-1,2-propanediol and 1,3-dichloro-2-
propanol, a modified form of the general monograph format has been
used, presenting separately the biological data for each.
3-CHLORO-1,2-PROPANEDIOL
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
3-chloro-1,2-propanediol was able to cross the blood-testis
barrier, blood-brain barrier and was distributed widely in body
fluids (Edwards et al. 1975). Accumulation of 3-chloro-1,2-
propanediol was seen in the cauda epididymis of rats and to a lesser
extent in mice through autoradiography (Crabo & Appelgren, 1972).
This finding was disputed by Jones et al., (1978), who did not
observe any tissue-specific retention of radioactivity in rats
injected intraperitoneally with 100 mg/kg bw 36C-labelled
3-chloro-1,2-propanediol. Neither 3-chloro-1,2-propanediol nor the
metabolite ▀-chlorolactate was accumulated in the tissue (Jones
et al., 1978).
A single intraperitoneal injection of 100 mg/kg bw of
14C-labelled 3-chloro-1,2-propanediol was given to male Wistar
rats. After 24-hours 30% of the dose was exhaled as 14CO2 and
8.5% was excreted unchanged in the urine (Jones, 1978). In another
study in rats which were injected intraperitoneally with a single
dose of 100 mg/kg bw 36C-labelled 3-chloro-1,2-propanediol, 23% of
the radioactivity was recovered in the urine as ▀-chlorolactate
(Jones et al., 1978).
2.1.2 Biotransformation
3-chloro-1,2-propanediol is detoxified by conjugation with
glutathione yielding S-(2,3-dihydroxypropyl)cysteine and the
corresponding mercapturic acid, N-acetyl-S-(2,3-dihydroxypropyl)
cysteine (Jones, 1975). 3-chloro-1,2-propanediol undergoes
oxidation to ▀-chlorolactic acid and further to oxalic acid (Jones
and Murcott, 1976). Formation of an intermediate metabolite,
▀-chlorolactaldehyde may also take place as traces of this substance
have been determined in the urine in rats (Jones et al., 1978).
The intermediate formation of an epoxide has been postulated, but
not proven (Jones, 1975).
2.1.3 Effects on enzymes and other biochemical parameters
The activity of all glycolytic enzymes in the epididymal and
testicular tissue was reduced in rats given daily subcutaneous
injection of 6.5 mg/kg bw/dy 3-chloro-1,2-propanediol for 9 days
(Kaur & Guraya, 1981a).
Ram sperm incubated with 3-chloro-1,2-propanediol has shown
that 3-chloro-1,2-propanediol inhibits the glycolysis of spermatozoa
in vitro (Brown-Woodman et al., 1975), possibly a result of
indirect inhibition of glyceraldehyde-3-phosphate dehydrogenase
(Suter et al., 1975; Mohri et al., 1975). Decrease in the
spermatozoa glycolytic enzymes was suggested to be a result of
altered epididymal milieu (Kuar & Guraya, 1981b).
Rats receiving daily doses of 6.5 mg/kg bw 3-chloro-1,2-
propanediol for a period of 9 days showed significantly decreased
(p&lt0.05) levels of RNA and protein in the testis and epididymis and
the observations were closely related to a parallel increase in the
concentration of proteinase and ribonuclease. The DNA content was
unchanged (Kaur & Guraya, 1981c).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
The oral LD50 of 3-chloro-1,2-propanediol was reported to be
152 mg/kg bw in rats (Ericsson & Baker, 1970).
2.2.2 Short-term toxicity studies
2.2.2.1 Rats
Groups of 8 male Fisher 344 rats were treated with a single
subcutaneous injection of 75 mg/kg bw 3-chloro-1,2-propanediol and
killed after 24 hours, 3, 8, 25, and 75 days, respectively. A
slight but significant (p&lt0.05) increase in liver weight was
observed after 24 hours while this finding was not found at later
sacrifices. Histologically the hepatocytes showed mild to moderate
cytoplasmatic swelling in the periportal area (Kluwe et al.,
1983).
Intraperitoneal injection of a single dose of 100 mg/kg bw
3-chloro-1,2-propanediol caused a increased diuresis for up to 15
days in male Sprague-Dawley rats. Higher doses (figure not
reported) caused anuresis and death, and histological examination of
the kidney showed acute glomerular nephritis. The type of kidney
lesions was characteristic of oxalic acid poisoning and crystals
characteristic of calcium oxalate were seen by microscopical
examination of the urine. Oral treatment with 10 mg/kg bw/dy
3-chloro-1,2-propanediol for five consecutive days did not cause any
increased diuresis in rats (Jones et al., 1978).
Another study showed that intraperitoneal injection of 100 and
120 mg/kg bw 3-chloro-1,2-propanediol caused severe proteinuria and
glucosuria in male Wistar rats. Oliguria and anuria were observed
and 4/9 animals died. The 5 surviving animals showed decreased
appetite and body weight, proteinuria, dose-related diuresis and
increased water intake (Morris and Williams, 1980).
Testing of (R)- and (S)-isomers of 3-chloro-1,2-propanediol,
synthesized under laboratory condition, has shown that only the
(R)-isomer induced a period of diuresis and glucosuria in rats
(Porter and Jones, 1982).
Oxalic acid, a metabolite of 3-chloro-1,2-propanediol, appeared
to play a important role in the development of kidney damage (Jones
et al., 1979). Birefringent crystals characteristic of calcium
oxalate present in tubules at the cortico-medullary junction were
early (1 day) morphological changes seen in rats treated with a
single subcutaneous injection of 75 mg/kg bw 3-chloro-1,2-
propanediol. On day 75 focal tubular necrosis, regeneration, and
tubular dilatation were observed in the kidney (Kluwe et al.,
1983).
Groups of 20 Sprague-Dawley rats of each sex were given 0, 30,
or 60 mg/kg bw/dy 3-chloro-1,2-propanediol by gavage 4 x 5 days over
a period of 4 weeks. 10 animals/group and sex were sacrificed on
day 2 and examined for clinical chemical parameters in the blood.
On day 2, rats of the high-dose group showed elevated activity of
serum glutamate-pyruvate-transaminase (males, p&lt0.05; females,
p&lt0.001), and elevated levels of creatinine (females, p&lt0.001),
urea and glucose (females, p&lt0.05). On day 25, treated rats
exhibited elevated activity of glutamate-pyruvate-transaminase
(males high-dose, p&lt0.001; females low and high-dose, p&lt0.001),
and elevated serum urea in high-dose males (p&lt0.001) and females
(p&lt0.05). Statistically significant (p&lt0.05 or lower) decreased
values of haemoglobin and haematocrit of treated male and female
rats were observed. Female rats in the high-dose group had
decreased erythrocyte count (p&lt0.001). Treated rats showed lowered
body weight gain, which at termination of the study was
statistically significant (statistics not reported). After 2 days
of treatment the relative organ weights of the kidney were elevated
(p&lt0.001), (males high-dose; females low and high-dose). On day 25
treated rats had significantly elevated relative weights of the
kidney, liver, and testis (males high-dose) (p&lt0.01 or 0.001).
Histopathological examination revealed chronic progressive
nephropathy of 8 females in the high-dose group, mild tubular
dilatation in the testis of 3 males in the low-dose group and 7 in
the high-dose group. One male in the high-dose group had severe
atrophy of both testes (Marchesini and Stalder, 1983).
Groups of 20 Fisher 344 rats of each sex were administered
3-chloro-1,2-propanediol in their drinking water at concentrations
of 0, 100, 300, or 500 mg/l over a period of 90 days. The exposure
corresponded to average daily intake levels of 9, 27 and 43 mg/kg bw
3-chloro-1,2-propanediol in males and 11, 31, and 46 mg/kg bw
3-chloro-1,2-propanediol in female rats. Ten animals of each
sex/group were sacrificed (interim sacrifice) after 30 days of
treatment. Clinical chemical and haematological parameters were
determined. Histopathological examinations were carried out on the
high-dose and control groups.
A slight anaemia (p&lt0.05 or 0.001) was evident in the middle-
and high-dose females after 30 days and in rats of both sexes after
90 days of treatment (p&lt0.05 or 0.01). However, no morphological
evidence of impaired haematopoiesis nor increased degradation of
erythrocytes were observed. A dose-dependent decrease (p&lt0.01) in
plasma creatinine of both sexes (middle and high-dose groups) was
seen after 30 days of treatment and at terminal sacrifice in all
treated groups (p&lt0.05 or 0.01). Serum phosphate levels in
high-dose male rats were increased at interim (p&lt0.01) and terminal
sacrifice (p&lt0.05). A statistically significant (p&lt0.01)
dose-dependent increase in relative organ weights was found for the
kidney and liver, and the increase of the relative kidney weight was
significant at the lowest dose level. Histopathological examination
of the high-dose and control groups revealed a lower incidence of
crystalline precipitations in the kidneys of treated animals
compared to the controls. In the livers of dosed rats, single
hepatocytes with 2-3 nuclei were noted in about half of the males
after 90 days of treatment. In the epididymis an increased number
of exfoliated spermatozoids of treated male rats was observed
(Marchesini et al., 1989).
2.2.2.2 Monkeys
Three out of 6 monkeys given 30 mg/kg bw 3-chloro-1,2-
propanediol perorally/day for 6 weeks showed haematological
abnormalities: anaemia, leukopenia and severe thrombocytopenia
(Kirton et al., 1970).
2.2.3 Long-term toxicity/carcinogenicity studies
2.2.3.1 Mice
A group of 50 female mice (CHR/Ha Swiss) was injected
subcutaneously with 1 mg 3-chloro-1,2-propanediol/mouse/week over a
period of 580 days. A second group of 50 mice was treated 3x/wk
with 2 mg 3-chloro-1,2-propanediol (dissolved in acetone)/mouse by
topical application. No changes were observed in the group treated
by dermal application. After subcutaneous application, local
sarcomas were found at the site of application in one dosed and one
control mouse (Van Duuren et al., 1974).
2.2.3.2 Rats
Three groups of 26 male and female Charles River CD rats
received 0, 30, or 60 mg 3-chloro-1,2-propanediol by gavage twice
weekly. After 10 weeks the doses were increased to 35 and 70 mg/kg
bw. The animals were treated for 72 weeks and the study was
terminated after 2 years. Three parathyroid adenomas were found in
male rats at the high-dose level. However, this finding was not
statistically significant when compared with the control group. The
authors did not find the result conclusive indication that
3-chloro-1,2-propanediol is a parathyroid carcinogen. While the
females showed no signs of toxicity, dosed male rats showed a higher
mortality. All male rats at both dose levels showed severe
testicular degeneration and atrophy (Weisburger et al., 1981).
Four groups of Fisher F344 rats (50 animals/sex/group, SPF
quality, 5-6 weeks old at start of the study, 11 days
acclimatization period prior to study initiation) received either 0,
20, 100, or 500 ppm 3-chloro-1,2-propanediol (equivalent to a mean
daily intake of 0, 1.1, 5.2, 28 mg/kg bw/day for males and 0, 1.4,
7.0, or 35 mg/kg bw/day for females) in their drinking water (tap
water) for a period of 104 weeks. Feed and tap water were provided
ad libitum. Feed was certified laboratory chow, feed contaminants
were within acceptable range according to EPA, USA. Test substance
was 3-chloro-1,2-propanediol, 98% pure, one batch used for the
entire study. Stability: more than 4 days in water, test solution
was prepared twice a week and tested once per group per week. Tap
water contaminants: a mean concentration of 2.7 ppm 3-chloro-1,2-
propanediol was determined (tested once/week). The report does not
comment on presence of 3-chloro-1,2-propanediol in provided water.
Experimental animals were examined daily for signs of ill
health or behavioural changes. Food consumption and body weight
were recorded weekly from start to week 19 (feed consumption) and
week 20 (body weight) of the study and thereafter monthly. From
week 88 to the end of the study, the body weight was recorded
weekly. Water consumption was recorded weekly from start to week 20
of the study, and thereafter fortnightly. Ophthalmological
examination was performed regularly. Haematological examination and
blood chemistry were performed on blood samples taken at day 722 to
737 from all surviving animals. All animals found dead or animals
killed "in extremis", as well as those killed at the end of the
experiment, were subjected to complete necropsies and
histopathological examination. The liver, spleen, pancreas, heart,
adrenals, testis, epididymides and brain were weighed.
The body weights were significantly (P&lt0.05) reduced in
high-dose male and female rats following the first week of
treatment. At termination the body weights were significantly
reduced (P&lt0.05 or lower) in intermediate-, and high-dose animals
showing a reduction in body weights of 33% (males) and 35% (females)
in high-dose rats. However, the mortality was unaffected by
treatment, and at terminal sacrifice more than 42% of the group
survived. The food and water intake were significantly (P&lt0.05)
reduced in high-dose male and female rats. No treatment-related
clinical signs were noted. The results of the haematological and
blood clinical chemical parameters varied considerably within the
groups, however no consistent significant dose-related effects were
observed. The reduced body weight in intermediate-, and high-dose
rats made it difficult to interpret a possible effect of treatment
on organ weights. However, the body weights were unaffected in
low-dose rats, of which the males showed significant (P&lt0.05)
increased kidney weight (absolute only).
Dose-related increased (or decreased) incidence of
hyperplasia/tumours were observed in the control, low- intermediate-
and high-dose groups in the following organs: Kidney: tubular
adenoma, males 0/50, 1/50, 1/50, 5/50, females 0/50, 1/50, 0/50,
9/50 (P&lt0.05). Tubular hyperplasia, males 3/50, 6/50, 15/50, 34/50
(P&lt0.05 in intermediate-, and high-dose when tubular adenoma and
tubular hyperplasia were combined), females 2/50, 4/50, 20/50, 31/50
(P&lt0.05). Testes: Leydig cell adenoma, 38/50, 43/50, 50/50
(P&lt0.001), 47/50 (P&lt0.05). Leydig cell adenocarcinoma, 0/50,
0/50, 0/50, 3/50 (P&lt0.05). Nodular Leydig cell hyperplasia was
present in a high proportion of controls and the incidence decreased
significantly in a dose-dependent pattern. The incidence was 39/50,
27/50, 4/50, 0/50. When nodular Leydig cell hyperplasias, adenomas
and carcinomas were combined for statistical analysis, there were no
significant difference between groups. Mammary gland (males):
fibroadenoma 0/50, 0/50, 2/50, 10/50 (P&lt0.01). Adenoma 0/50, 0/50,
1/50, 1/50. Adenocarcinoma 0/50, 0/50, 1/50, 1/50. Preputial
gland: adenoma 1/50, 2/50, 6/50 (P&lt0.05), 5/50. Carcinoma 0/50,
0/50, 1/50, 2/50 (P&lt0.05). When adenomas and carcinomas were
combined for statistical analysis, the resulting increased incidence
was significant for both intermediate-, and high-dose groups.
Pancreas: There was a treatment-related decrease in the incidence of
islet-cell hyperplasias, adenomas, and carcinomas in male rats. The
incidences were for islet-cell hyperplasia 14/50, 8/50, 5/50, 1/50.
Islet-cell adenoma 16/50, 9/50, 7/50, 0/50. Islet-cell carcinoma
8/50, 0/50, 2/50, 0/50. When hyperplasias and neoplastic lesions
were combined for statistical analysis, the decrease in incidence
was significant at all dose levels (P&lt0.05 or lower). Chronic
progressive nephropathy occurred in both sexes in all groups and the
incidence increased with dose being significant at the
intermediate-, and high-dose level (P&lt0.05 or lower). Female rats
were more severely affected than males. The figures were 36/50,
40/50, 45/50, 49/50 (males) and 24/50, 23/50, 42/50, 48/50
(females). Correlations (P&lt0.001) between the severity of the
nephropathy and the kidney tubular hyperplasia and kidney adenoma
were found to be significant (P&lt0.01).
A dose-dependent increase in epithelial single cell
degeneration was observed in the epididymis. The incidence was
significant at intermediate-, and high-dose level (P&lt0.001).
The report concludes that treatment with 3-chloro-1,2-
propanediol caused increases in renal and testicular Leydig cell
tumours. Renal tumours developed in a dose-dependent fashion in
both sexes and were considered secondary to the 3-chloro-1,2-
propanediol treatment-related increase in chronic progressive
nephropathy. The treatment-related increase and acceleration of
Leydig cell tumours may be considered as hormone-mediated promotion.
3-chloro-1,2-propanediol treatment caused a dose-related increase in
mammary and preputial gland tumours in the males. This effect may
be considered as secondary to hormonal activity of large Leydig cell
tumours (Sunahare et al, 1993).
2.2.4 Reproduction studies
3-Chloro-1,2-propanediol has been reported to exert an
inhibitory activity on male fertility (Gunn et al., 1969; Helal,
1982) and the effect is reversible (Ericsson & Youngdale, 1970;
Jones, 1983). The mechanism of the antifertility activity of
3-chloro-1,2-propanediol is not known in detail. However, it has
been shown that the metabolites of 3-chloro-1,2-propanediol have an
inhibitory activity on enzymes in spermatozoa glycolysis, resulting
in a reduced motility of the spermatozoa (Jones, 1983). Inhibition
of spermatozoa motility was suggested partly to be due to alkylation
of spermatozoa cysteine by 3-chloro-1,2-propanediol (Kalla & Bansal,
1977). 3-Chloro-1,2-propanediol also affects several enzymes of
epithelial cells in the testis and caput epididymis, resulting in
decreased glycolysis (Gill & Guraya, 1980). It is suggested that
only the 3-chloro-1,2-propanediol (S)-isomer, synthesized under
laboratory condition, possesses a specific inhibitory action on
glycolysis in boar sperm (Stevenson and Jones, 1984).
3-Chloro-1,2-propanediol has two specific effects on the
reproductive tract of the male rat. These effects were dose-
dependent and have been classified as the high-dose effect and the
low-dose effect. The high-dose effect followed a single
intraperitoneal injection of 75 mg/kg bw 3-chloro-1,2-propanediol.
Bilateral retention cysts or spermatocele of the caput epididymis
developed 5 to 7 days after treatment (Cooper & Jackson, 1973).
Studies using electron microscopy have shown, that 3-chloro-1,2-
propanediol, given by gavage at a level of 140 mg/kg bw,
specifically affected the epithelia localized in the initial segment
of epididymis in male rats 2 hours later. The cellular lesions were
characterised by sloughing of the epithelium, which led to
obstruction of the epididymal tract (Hoffer et al., 1973). The
back-pressure of the testicular fluid caused oedema, inhibition of
spermatogenesis and atrophy of the testis (Jones, 1983).
Histological examination of testes from rats treated with daily
injection of 40 mg/kg bw 3-chloro-1,2-propanediol for 20 days
revealed total inhibition of the spermiogenesis by presence of
degeneration and disappearance of the spermiogonia from the tubules.
Proliferation of the epithelial cells of the ducts in the cauda
epididymis was observed and several blood vessels showed thickened
walls (Samojlik and Chang 1970).
The low-dose effect was directed towards mature sperm contained
in the cauda epididymis. The effect, which was evident after a few
days following oral treatment of rats with levels of 5-10 mg/kg bw
3-chloro-1,2-propanediol/dy, rendered the spermatozoa incapable of
fertilization without causing any visible changes in their
morphology (Jones 1983). Male rats treated with daily subcutaneous
injections of 15 or 40 mg/kg bw 3-chloro-1,2-propanediol showed
infertility 6 and 3 days after commencement of treatment,
respectively. If the treatment with 15 mg/kg bw 3-chloro-1,2-
propanediol was continued for 30 days recovery of fertility was
observed 18 days after cessation of the treatment (Samojlik and
Chang, 1970). The lowest doses shown to cause infertility of male
rats, determined by mating studies, were observed at the following
daily orally treatment of male rats with 3-chloro-1,2-propanediol:
6.5 mg/kg bw for 10 days (Gunn et al., 1969); 5 mg/kg bw for 14
days (Coppola, 1969); 2.5 mg/kg bw at "continuous" treatment
(Erickson & Bennett, 1971); (subcutaneous injection): 8 mg/kg bw for
3 days (Black et al., 1975); 8 mg/kg bw for 4 days (Turner, 1971).
Groups of 5 albino male rats treated perorally for 10 to 12
days with either 0.5, 1.0, 2.0, 4.0, or 6.0 mg/kg bw 3-chloro-1,2-
propanediol showed 2.5%, 20%, 45%, 85% and 100% sterility (sterility
was based upon histological degree of spermiogenesis), respectively
(Helal, 1982).
The following abstract has been compiled from a summary report:
groups of 5 Wistar male rats were dosed with 0 (distilled water),
0.1, 0.5, 1, 2, 3, 4, 5, or 10 mg/kg bw/dy 3-chloro-1,2-propanediol
by gavage for 7 days prior to, and during mating. Each male rat was
mated with a total of 5 virgin females which were sacrificed on day
14 of gestation and examined for pregnancy status. 3-Chloro-1,2-
propanediol induced no adverse effect on male fertility at a dose
level of 3 mg/kg bw/dy and lower as shown by the pregnancy rate,
total implantations and number of live embryos. However, the
pre-implantation loss was significantly greater (p=0.05) for female
rats mated with males given 3 mg/kg bw/dy 3-chloro-1,2-propanediol
when compared to controls. The NOEL was 2 mg/kg bw/dy (Parish,
1989).
Antifertility activity of 3-chloro-1,2-propanediol in other
species than rat has been reported in males of hamster, gerbil,
guinea pig, dog, ram and rhesus monkey in vivo (Jones, 1983).
3-chloro-1,2-propanediol was reported to have no antifertility
activity in the mouse, quail or rabbit (Jones, 1978).
Groups of 10 female rats were injected subcutaneously with 0 or
10 mg 3-chloro-1,2-propanediol (approx. 25 mg/kg bw) every second
day for a period of 30 days. Significant (p&lt0.01) decrease was
noted in the relative organ weights of the ovary, uterus and vagina
of treated females compared to the controls. Histological
examination revealed the following changes of the treated female
rats: the ovary appeared small in size and showed wide spread
follicular atresia and degeneration of corpora lutea; in the uterus
the gland was regressed and the lumen was lined with columnar
epithelium; atrophic changes were observed in the vaginal
epithelium. The protein and RNA content in the uterus and vagina
were significantly (p&lt0.01) reduced in the treated females compared
to controls. The authors suggested a luteolytic and possibly
antioestrogenic effect of 3-chloro-1,2-propanediol in female rats
(Lohika and Arya, 1979).
2.2.8 Special studies on genotoxicity
The results of genotoxicity studies with 3-chloro-1,2-
propanediol are summarized in Table 1.
2.3 Observations in humans
A synergistic effect of 3-chloro-1,2-propanediol and copper
ions in decreasing the motility of human spermatozoa was observed
in vitro (Kalla & Singh, 1981). When 3-chloro-1,2-propanediol was
incubated with ejaculated human sperm the motility of the
spermatozoa was inhibited and their metabolic activity was reduced,
as measured by glucose, oxygen uptake and lactate production
(Homonnai et al., 1975).
Table 1. Results of genotoxicity tests on 3-chloro-1,2-propanediol
Test system Test object Concentration of +/- Reference
3-chloro-1,2-propanediol
In vitro bacterial S.typhimurium TA1535, 2-200 Ámol/plate + (2) Silhankova et al., 1982
mutagenicity assay (1) TA1537, TA1538, TA98
S.typhimurium TA100 10-1 000Ámol/plate + Stolzenberg & Hine, 1980
E.coli TM930 2-200 Ámol/plate - Silhankova et al., 1982
Forward-mutation Schizosaccharomyces 100-300 mM + Rossi et al., 1983
assay on yeast(1) plombe
Mammalian cell mutation Mouse lymphoma TK 2-9 mg/ml + (5) Henderson et al., 1987
assay (1) locus assay
Mammalian cell mutation HeLa cell (6) - Painter & Howard, 1982
assay (1)
Mammalian cell mutation Mouse fibroblast 0.1-2 mg/ml + Piasecki et al., 1990
assay M2-clone
Sister chromatid exchange Chinese hamster 700-2800 Ág/ml + May, 1991
assay(1) V79 cells
Mammalian cell HPRT-test Chinese hamster 0.3-70mM ? (4) G÷rlitz, 1991
(1) V79 cells
Table 1 (contd).
Test system Test object Concentration of +/- Reference
3-chloro-1,2-propanediol
In vivo dominant lethal ICR/Ha Swiss mice (3) - Epstein et al., 1972
assay
Micronucleus test OF1 mice 40-120 mg/kg bw - Jaccaud & Aeschbacher, 1989
(1) with and without metabolic activation
(2) no frame shift mutations in strains TA1537, TA1538 or TA98
(3) single intraperitoneal injection of 125 mg/kg bw 3-chloro-1,2-propanediol or peroral treatment of 20 mg/kg bw
3-chloro-1,2-propanediol for five days
(4) weak mutagenic effect only at toxic dose level (50mM)
(5) positive only after metabolic activation
(6) not reported
1,3-DICHLORO-2-PROPANOL
2. Biological data
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
No information was available.
2.1.2 Biotransformation
▀-Chlorolactate (approx. 5% of dose), N,N'-bis-acetyl-S,S'-
(1,3-bis-cysteinyl)propan-2-ol (approx. 1% of dose), and N-acetyl-S-
(2,3-dihydroxypropyl)cysteine were identified in the urine of rats
treated orally with 50 mg/kg bw/dy 1,3-dichloro-2-propanol for 5
days. The authors proposed that epoxy-halopropane ( epi-
chlorohydrin) is formed as an intermediate, which may either undergo
conjugation with glutathione to form mercapturic acid or be
hydrolyzed to 3-chloro-1,2-propanediol. The latter undergoes
oxidation to ▀-chlorolactate which is further oxidized to oxalic
acid. Formation of other epoxides was postulated. However, the
formation of epoxides from alpha-chlorohydrins only takes place at
high pH-values and is unlikely to occur under physiological
conditions (Jones and Fakhouri, 1979).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
The oral LD50 of 1,3-dichloro-2-propanol was reported to be
122 mg/kg bw in rats, while by intraperitoneal application the LD50
was 106 mg/kg bw (Pallade et al., 1963). In rabbits the LD50 was
800 mg/kg bw following dermal application (Smyth et al., 1962).
In mice the LC50 over a period of 1-15 days was 1.7-3.2 mg/l air
(Pallade et al., 1963). When tested on rabbit eyes 1,3-dichloro-
2-propanol caused irritation and moderately severe damage (Grant,
1974).
2.2.2 Short-term toxicity studies
2.2.2.1 Rats
The following summary was written from an abstract cited in
The Toxicologist. A critical evaluation of the findings from this
abstract has not been possible. 1,3-dichloro-2-propanol was
evaluated for subchronic toxicity in Sprague-Dawley rats
(10/sex/dose group) treated with dose levels of 0, 0.1, 1, 10, or
100 mg/kg bw/dy 1,3-dichloro-2-propanol by gavage in distilled water
5 days/week for 13 weeks. Decreases in bw gain, feed consumption
and haematologic parameters, increases in liver and kidney weights,
alterations in serum chemistry and urinary parameters, gross
pathologic changes in the stomach and histopathologic changes in the
stomach, kidney, liver and nasal tissue were observed at 100
mg/kg/day in males and females. The changes in serum chemistry were
considered secondary to renal and hepatic changes observed in high-
dose animals. At 10 mg/kg, increased liver weights in males and
females and histopathologic changes in the stomach, kidneys and
liver in males were observed. The treatment related-effects
observed at 10 mg/kg were less frequent and/or less severe than the
effects observed at 100 mg/kg. No effects were observed at 0.1 or 1
mg/kg in males or females (Jersey et al., 1991).
2.2.3 Long-term toxicity/carcinogenicity studies
2.2.3.1 Rats
In a combined long-term toxicity/carcinogenicity study, 4
groups of 80 male and 80 female rats (Wistar KFM/Han, initial age of
4 weeks; 10 days acclimatization prior to test), received
1,3-dichloro-2-propanol [purity: 99%; stability confirmed by sponsor
at six-month intervals] in their drinking water over a period of up
to 104 weeks. 1,3-dichloro-2-propanol concentrations in the
drinking water (daily preparation of 1,3-dichloro-2-propanol/water
mixture, regular determination of 1,3-dichloro-2-propanol stability,
concentration and homogeneity) were 0, 27, 80, or 240 mg/l
corresponding to intakes of 0, 2.1, 6.3, and 19.3 mg/kg bw/day for
male rats and 0, 3.4, 9.6, and 30 mg/kg bw/day for female rats. The
diet [pelleted; regular determination of contaminants showed
presence of low, biologically insignificant levels of aflatoxin,
estrogen, pesticides and heavy metals] was provided ad libitum.
Interim kill was performed on 10 rats of each sex and group after
26, 52, and 78 weeks of treatment.
Haematologically, female rats in the high-dose group, in
particular, showed statistically significantly (p&lt0.05) decreased
haemoglobin concentration and haematocrit (26 and 104 weeks), and
red blood cell count (104 weeks). Clinical biochemical and urine
analysis findings suggested hepatotoxicity primarily in high-dose
females. Statistically significant (p&lt0.05) increased activity of
aspartate- and alanine aminotransferase (78 and 104 weeks), alkaline
phosphatase (104 weeks), and gamma-glutamyltransferase (104 weeks)
were observed in female rats. Statistically significant (p&lt0.05)
increases in urinary levels of protein and amylase were noted in
high-dose female rats after 52, 78, and 104 weeks of treatment.
Increased mortality was observed in high-dose males (32/50) and
females (27/50) compared to that in the controls (males 18/50;
females 13/50), (statistics not reported). The mortality of the
low-dose group was: 11/50 (males), 9/50 (females); of the
intermediate-dose group was: 16/50 (males), 14/50 (females).
There were no treatment-related signs of toxicity nor changes
in food and water consumption. However, statistically significant
(p&lt0.05 or lower) reductions in mean body weights were observed in
high-dose males after 74 weeks and in high-dose females after 78
weeks. A dose-related increase in the relative organ weights was
observed in a number of organs, in particular, the liver and kidney.
After 26 weeks: liver of males and females in all treated groups
(p&lt0.05); kidney of males at intermediate- and high-dose (p&lt0.05),
and females at high-dose (p&lt0.05). After 52 weeks: livers of males
and females in intermediate- and high-dose groups (p&lt0.05); kidney
of females at high-dose (p&lt0.05). After 78 weeks: liver and kidney
of males and females at high-dose (p&lt0.01). After 104 weeks:
liver, kidney and brain of males and females at high-dose (p&lt0.01).
Histopathological examination revealed occurrence of several tumours
in various organs. Among these tumours dose-related neoplastic
lesions in middle- and high-dose male and female rats were seen.
Statistically significant positive trends were found for
hepatocellular adenoma (females, p&lt0.001); hepatocellular carcinoma
(males and females, p&lt0.001); hepatic hemangiosarcoma (males,
p&lt0.01 and females, p&lt0.05); renal tubular adenoma (males,
p&lt0.001); renal tubular carcinoma (males, p&lt0.05); lingual
papilloma (males and females, p&lt0.001); lingual papillary carcinoma
(males, p&lt0.001 and females, p&lt0.01); thyroid follicular adenoma
(females, p&lt0.05); thyroid follicular carcinoma (males, p&lt0.01).
These neoplastic lesions occurred in treated animals after 26 weeks
(hepatocellular adenoma), 52 weeks (hepatocellular adenoma and
carcinoma, lingual papilloma and carcinoma), and 78 weeks
(hepatocellular carcinoma, renal tubular adenoma, lingual papilloma
and carcinoma, thyroid follicular adenoma). In addition to the
above-mentioned tumours, one stomach papilloma was found in one
high-dose female rat after 78 weeks and at terminal sacrifice one
stomach carcinoma (low-dose, female), carcinomas in the oral cavity
[intermediate-dose (one, female) and high-dose (two, males)].
The incidence of the above-mentioned neoplastic lesions in
control rats was: two hepatocellular adenomas (male and female) and
one thyroid follicular adenoma (female). Among non-neoplastic
lesions the liver showed dose-dependent increase in incidence of
fatty change, eosinophilic foci, glycogen free foci, Kupffer cell
haemosiderin storage, and peliosis. Follicular hyperplasia was
evident in thyroid glands of high-dose males. These results
strongly suggest an oncogenic effect of 1,3-dichloro-2-propanol on
liver, kidney, oral epithelia/tongue and thyroid gland in rats at
the intermediate- and high-dose level. The significance of the
sinusoidal peliosis observed in all treated groups was not clear.
However, peliosis has been suggested to represent a pre-neoplastic
stage of vascular hepatic neoplasia (Wayss et al., 1979).
The increased incidence of hepatic fatty change and
haemosiderin-storing Kupffer cells in the liver in animals in the
intermediate- and high-dose groups were suggested to reflect a
metabolic disturbance of the liver caused by 1,3-dichloro-2-propanol
(RCC, 1986).
2.2.4 Reproduction studies
2.2.4.1 Rats
The following summary has been obtained from an abstract cited
in Hazardous Substances Data Base. A critical evaluation of
material from this abstract has not been possible: Groups of 20, 10
or 10 male Wistar rats were dosed with either water (controls), 15,
or 60 mg/kg bw/dy 1,3-dichloro-2-propanol by gavage for 14 days,
respectively. Treated rats showed appearance of spermatocele or
sperm granuloma formation in the epididymides (Tunstall
Laboratories, 1979).
Investigations on the genotoxic mechanisms of 1,3-dichloro-2-
propanol (Hahn et al., 1991), indicate that the genotoxic effect
of 1,3-dichloro-2-propanol depends on the chemical formation of
epichlorohydrin, which has mutagenic activity (Rossi et al.,
1983).
2.2.8 Special studies on genotoxicity
Table 2. Results of genotoxicity tests on 1,3-dichloro-2-propanol
Test system Test object Concentration +/- Reference
1,3-dichloro-2-propanol
In vitro Bacterial S.typhimurium TA1535, 2-200 Ámol/plate + (2) Silhankova, et al., 1982
mutagenicity assay (1) TA1537, TA1538, TA98
S.typhimurium TA100 0.1-10Ámol/plate + Stolzenberg & Hine, 1980
S.typhimurium TA100, 3-300Ámol/plate + Nakamura et al., 1979
TA1535
E.coli, TM930 2-200Ámol/plate + (3) Silhankova et al., 1982
Mammalian cell mutation Mouse lymphoma TK 2-9 mg/ml + Henderson et al., 1987
assay(1) locus assay
Sister chromatid exchange Chinese hamster V79 0.12-3.3 mM + (5) Von der Hude et al., 1987
assay(1) cells
Mammalian cell mutation HeLa cell 2.5x103 M (4) + Painter & Howard, 1982
assay(6)
Mammalian cell mutation Mouse fibroblast 0.1-1 mg/ml + Piasecki et al., 1990
assay M2-clone
(1) with and without metabolic activation
(2) no frame shift mutations in strains TA1537, TA1538 or TA98
(3) only positive after metabolic activation
(4) effective concentration
(5) almost inactivated with metabolic activation
(6) only tested with metabolic activation
2.3 Observations in humans
Severe irritation of the throat and stomach has been described
as a likely effect after ingestion of 1,3-dichloro-2-propanol
(Gosselin et al., 1976).
3. COMMENTS
3-Chloro-1,2-propanediol
3-Chloro-1,2-propanediol has been shown to increase the
relative kidney weights of rats treated for 4 weeks (30 mg/kg bw/dy
by gavage), or 3 months (9 mg/kg bw/dy in the drinking water) and
absolute kidney weights when treated for 104 weeks (1.1 mg/kg bw/dy
in the drinking water). A single subcutaneous injection of 75 mg
3-chloro-1,2-propanediol/kg bw to rats caused renal tubular necrosis
and dilatation. A no-effect level for the effect on the kidney was
not observed.
In monkeys 3-chloro-1,2-propanediol induced anaemia,
leucopenia, and thrombocytopenia following ingestion of 30 mg/kg
bw/dy for 6 weeks.
Data presented to the Committee clearly demonstrated that
3-chloro-1,2-propanediol possesses an inhibitory effect on male
fertility in rats and that the effect is reversible. This effect is
caused by an inhibition of glycolytic enzymes in the epididymis,
testicular tissue, and in spermatozoa, resulting in reduced motility
of the spermatozoa. No visible morphological changes of the
spermatozoa or epididymis were seen at dose levels of 5-10 mg
3-chloro-1,2-propanediol/kg bw/dy, while a single intraperitoneal
injection of 75 mg/kg bw caused development of retention cysts or
spermatocele of the caput epididymis in rats. In a reproduction
study the NOEL for male rat fertility was 2 mg/kg bw/dy when the
rats were treated orally with 3-chloro-1,2-propanediol for 7 days
and during the mating period.
3-Chloro-1,2-propanediol was genotoxic in most in vitro
assays, while it was negative in in vivo assays. In addition,
3-chloro-1,2-propanediol induced malignant transformation of mouse
M2-fibroblasts in culture.
The results of a recently-completed long-term
toxicity/carcinogenicity study in rats treated at dose levels of
1.1, 5.2 or 28 mg 3-chloro-1,2-propanediol/kg bw/dy in drinking-
water for 104 weeks indicated a carcinogenic effect. Occurrence of
treatment-related increased incidences of tumours in the kidneys of
both sexes and testis, mammary and preputial gland of male rats were
reported. Although it has been suggested that the occurrence of
these tumours might be secondary to either a sustained organ
toxicity (kidney) or hormonal disturbances (testis and mammary
gland), information was not available to the Committee to support
this assumption. The Committee noted that the drinking-water of the
control animals contained low levels of 3-chloro-1,2-propanediol.
The presence of 3-chloro-1,2-propanediol in the drinking-water may
have confounded the quantitative evaluation of the dose-response
relationships for carcinogenicity. In addition, significantly
increased kidney weights were observed in male rats at the lowest
dose level.
1,3-dichloro-2-propanol.
The Committee reviewed studies on biotransformation, acute
toxicity and long-term toxicity/carcinogenicity in rats, and
in vitro genotoxicity of 1,3-dichloro-2-propanol.
The results of a long-term toxicity/carcinogenicity study in
rats treated at dose levels of 2.1, 6.3, or 19 mg 1,3-dichloro-2-
propanol/kg bw/dy in the drinking-water for 104 weeks indicated a
carcinogenic effect of 1,3-dichloro-2-propanol. Induction of benign
and malignant tumours of the liver, kidney, thyroid gland, and oral
epithelia/tongue was observed in rats at the mid- and high-dose
levels.
1,3-Dichloro-2-propanol was active in a range of genotoxicity
screening assays, including tests for chromosomal effects in
mammalian cells in culture and tests for gene mutations in bacteria.
In addition, 3-chloro-1,2-propanediol induced malignant
transformation of mouse M2-fibroblasts in culture.
The Committee was not presented with results from studies on
absorption, distribution or excretion of 1,3-dichloro-2-propanol.
The Committee noted that different rat strains were used in the
long-term toxicity/carcinogenicity studies on 3-chloro-1,2-
propanediol and on 1,3-dichloro-2-propanol, which precluded a direct
comparison between these two compounds in regard to their
carcinogenicity.
4. EVALUATION
The Committee concluded that 3-chloro-1,2-propanediol and
1,3-dichloro-2-propanol are undesirable contaminants in food and
expressed the opinion that their levels in hydrolyzed vegetable
proteins should be reduced to the lowest technologically achievable.
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